1. Field of Invention
This patent provides materials and methods for the qualitative and quantitative assessment of ubiquitin and ubiquitin-like proteolytic enzyme activity as well as the discovery of novel enzymes, for evaluating and/or screening compounds for their effects on proteolytic enzyme activity, and for detecting activity in biological samples, which is useful for the diagnosis of conditions and diseases associated with altered enzyme levels, amounts, sequences and/or activities. This technology is also incorporated into disease models in the form of transgenic cells, plants and animals.
2. Background of the Invention
Ubiquitin (Ub) isopeptidases were first cloned over a decade ago. Up to this point, however, there existed no suitable assay for proteolytic enzymes specific for Ub or Ubiquitin-like Proteins (UBL) or for rapid screening of modulators or inhibitors of the enzyme. Most of the assays that are currently in use rely on cleavage of linear Ub-fusions, which are either produced in E. coli, e.g. tetra-Ub, Ub-CEP52, Ub-GSTP1, Ub-DHFR, Ub-PESTc, and the like, or synthesized chemically. In these assays the reaction products are either analyzed by gel electrophoresis, or selectively precipitated and then analyzed by liquid scintillation spectrometry.
These assays have significant drawbacks, e.g. that gel-based approaches are labor intensive and expensive. Although selective precipitation/scintillation count provides quantitative results and allows the processing of larger numbers of samples than gel-based assays, it requires centrifugation, and supernatant separation. Ubiquitin-7-amido-4-methylcoumarin (Ub-AMC) is a fluorogenic substrate for High Throughput Screening (HTS) that is commercially available and easy to use. However, unlike Ubiquitin C-terminal Hydrolases (UCHs), most Ubiquitin Specific Proteases (USPs) do not cleave small groups from the Ubiquitin (Ub) molecule. AMC, in addition, is highly hydrophobic and, based on its own interactions with test compounds, may give rise to false positives in screenings. Other ways to detect cleavage, e.g. High Pressure Liquid Chromatography (HPLC) and mass-spectroscopy have also been used although they have their own drawbacks. Furthermore, none of the prior art methods are suitable or adaptable to high throughput screening, which requires simple (minimal number of steps) assays that may be conducted using multi-well plates, and whose endpoints are read directly from the plates.
There is, therefore, a need for assays and kits that are simple and relatively inexpensive in nature while at the same time being suitable for conducting high throughput screening for modulators of ubiquitin or Ubiquitin-like Protein (UBL) proteolytic enzymes.